Papaverine

Arterioles were pre-constricted with endothelin-1 (Peninsula, San Carlos, CA, USA) and those that did not constrict beyond 30% were excluded from the study. The steady-state internal diameter was measured before and during intraluminal flow corresponding to pressure gradients of 10–100 cmH₂O or log increases in the concentration of acetylcholine (ACh; 10⁻⁹–10⁻⁴ M) [28 (link)]. This approach was repeated after incubating vessels with the NOS (nitric oxide synthase) inhibitor (L-NAME; 10⁻⁴ M), or the cyclooxygenase enzyme inhibitor, indomethacin (10⁻⁵ M) for 30 min. When possible, one arteriole from each biopsy was used for the whole protocol (baseline, LNAME, and indomethacin) measurements. However, if an arteriole did not constrict or dilate properly in response to endothelin-1 and papaverine, respectively, another arteriole from the same adipose tissue biopsy was used. At the end of each experiment, papaverine (10⁻⁴ M) was used to detect maximal vasodilation. The reported percentage of vasodilation was calculated as the % increase in diameters after each treatment condition relative to the pre-constricted state.

In this preparative scale fermentation, 40 flasks (500 mL each), each containing 100 mL of a stage II culture, were used. A solution of papaverine in DMF (400 mg/10 mL) was distributed amongst these flasks. In total, 850 mg of fermentation residue was recovered after 12 days. Using the S1 solvent system, two spots were located on Merck TLC sheets of silica gel (R_f = 0.9, 0.64). The residue was loaded on top of a silica gel column (90 g, 120 cm × 2.5 cm) after being solubilized in 2 mL of a MeOH-CH₂Cl₂ mixture (1:1). The ethyl acetate was used as the mobile phase for the gradient elution procedure, and the polarity was then increased with methanol in 5% increments of up to 80% methanol; 25 mL fractions were collected. Fractions (40–66) and (70–85) were pooled and evaporated to produce 20 mg and 25 mg of impure 3 and impure 4 residue, respectively. Column chromatography on Sephadex LH-20 (5 g, 60 cm × 1 cm) was used to further purify these compounds via elution with 100% methanol. Thirty fractions, each containing 5 mL, were collected. Pure compound 3 was produced via combining and evaporating fractions 15–20. This produced a white, amorphous powder, with R_f = 0.9 in S1, that was soluble in chloroform (5 mg, yield: 1.25%). For additional purification of 4, the same Sephadex LH-20 column (5 g, 60 cm × 1 cm) and 100% methanol elution were used. Twenty-five fractions, each containing 5 mL, were collected. Fractions (16–22) were combined and evaporated to give pure compound 4 as a white, amorphous powder that was soluble in chloroform (6 mg, 1.5% yield, R_f 0.64, S1).

Forty flasks of similar size (500 mL), each containing a stage II culture (100 mL), each received 400 mg of papaverine in 20 mL of DMF. In total, 800 mg of fermentation residue was recovered after 15 days of fermentation. The residue was loaded on top of a silica gel column (90 g, 120 cm × 2.5 cm) after being dissolved in a CH₃OH-CH₂Cl₂ mixture (1:1) (2 mL). Ethyl acetate was used as the mobile phase for the gradient elution procedure. The polarity was gradually increased via adding methanol in increments of 2%, of up to 80% methanol; 25 mL fractions were collected. Fractions (30–36) were collected and evaporated to produce impure 5 residue. Further purification was carried out using the Sephadex LH-20 column (5 g, 60 cm × 1 cm), which was eluted with 100% methanol. White, amorphous powder made up pure compound 5, which had an R_f value of 0.35 using S1. Metabolite 5 was soluble in chloroform (6 mg, 1.5% yield).